No abstract

When targeting a certain class of analytes, such as the phosphorylated lipids in complex biological extracts, interfering species can pose challenges to qualitative and quantitative analyses. Two aspects of lipid analysis were optimized to simplify the isolation and characterization of phosphorylated lipids in biological extracts. A new solid ionic crystal MALDI matrix was synthesized which combined the lipid response enhancing UV-absorber p-nitroaniline with the protonating agent butyric acid. Mass spectra of the extracts containing phosphorylated lipids were simplified by revealing only protonated molecules [M + H] + of the zwitterionic phosphatidylcholine (PC) headgroup-containing lipids, such as lyso-PC, PC, and platelet-activating factor. For the anionic phosphorylated lipids, such as phosphatidylglycerol, phosphatidic acid, and phosphatidylserine, further spectrum simplification is obtained by the appearance of only the monosodium adducts [M + Na] + as the major molecular ions, in preference to the double sodium adducts [M + 2Na -H] + . In addition, a new extraction, isolation, and cleanup procedure has been developed to prepare the phosphorylated lipids for MALDI-TOF analysis by the use of immobilized metal ion affinity chromatography media (i.e., ZipTip). The latter procedure was successfully applied to a complex biological tear film lipid layer extract in preparation for MALDI-TOF analysis and phospholipid characterization.Lipids are important biomolecules that are found in all living species. These include nonpolar lipids, such as the acylglycerols, and the more polar phosphorylated lipids. Of the nonpolar lipids, triacylglycerols are thought to be a storage form of energy in the cells, 1 whereas diacylglycerols are of special importance for their role as physiological activators of protein kinase C (PKC). 2,3 Of the polar lipids, those containing phosphoryl headgroups, such as the phosphatidylcholines (PC), the phosphatidylethanol-amines (PE), and the phosphatidylserines (PS) (see Figure 1 for structures), constitute the bilayer components of biological membranes, determine the physical properties of these membranes, and directly participate in membrane protein regulation and function. Other types of phosphorylated lipids are the sphingomyelins and the glycosphingolipids, which have been reported to be involved in different biological processes such as growth and morphogenesis of cells. 4 Thus, analysis of this special class of biomolecules has been of great interest to medical researchers, biologists, and chemists. In this paper, a MALDI-TOF MS study of the phosphorylated lipids of normal human eye tear is reported using a novel solid ionic crystal MALDI matrix consisting of p-nitroaniline (PNA) and butyric acid. MALDI-TOF has become an important tool for the characterization of complex biological extracts due to its high sensitivity, high resolution, and limited fragmentation of the compounds of interest. Front-end isolation of the phosphorylated lipids was achieved using immobilized metal a...

Millions of individuals suffer from a health condition known as keratoconjunctivitis sicca (KCS, also known as 'dry eye'). Studies have indicated that the lipids in the tear film layer, which covers the outer portion of the eye, may be directly correlated with the existence of dry eye syndrome. By identifying and comparing the major, non-polar lipids in normal eye tears with a dry eye model, it may be possible to identify a symptom of, or a contributing factor to, dry eye. Electrospray tandem mass spectrometry (ES-MS/MS) was used to identify and compare the non-polar lipids, detected as lithium adducts, from normal and dry eye tear samples obtained from rabbits. A limited number of normal human tear samples were also examined for lipid content, and a close resemblance to rabbit was observed. Three distinct regions were delineated in the ES mass spectra of the non polar lipids, m/z 20-500, 500-800 and 800-1100. A common feature noted among identified lipid components was a glycerol backbone with fatty acyl substituents attached. Product ion spectra were obtained for lithiated monoacyl-, 1,2-and 1,3-diacyl-and triacylglyceride standards. Newly proposed structures and fragmentation pathways for the major product ions are presented for the 1,2-and 1,3-diglycerides, and also for the monoglyceride. New approaches to distinguishing asymmetric 1,2-diglycerides and 1,2-from 1,3-diglycerides are proposed. For the rabbit tear samples, the m/z 20-500 range contains monoester diols with empirical formulas C n H 2n O 4 , the m/z 500-800 range includes diesters with empirical formulas C n H 2n−2 O 5 and the m/z 800-1100 range contains triesters with empirical formulas C n H 2n−4 O 6 . Also found in the extracts were three isoprene acetals (terpenoids).

In this study, we investigated the corneal epithelial cell growth rate and adhesion to novel hydrogels with (1) extracellular matrix proteins [fibronectin, laminin, substance P, and insulin-like growth factor-1 (IGF-1)] and (2) peptide sequences [RGD and fibronectin adhesion-promoting peptide (FAP)] tethered to their surface on poly(ethylene glycol) (PEG) chains. The growth rate to confluence of primary rabbit cornea epithelial cells was compared for plain polymethacrylic acid-co-hydroxyethyl methacrylate (PHEMA/MAA) hydrogels, PHEMA/MAA hydrogels coated with extracellular matrix proteins or peptides, and PHEMA/ MAA hydrogels with tethered extracellular matrix proteins or peptides on the surface. The development of focal adhesions by the epithelial cells grown on the surfaces was determined by F-actin staining. Little to no epithelial cell growth occurred on the plain hydrogel surfaces throughout the 15-day culture period. Of the coated hydrogels, only the fibronectin-coated surfaces showed a significant increase in cell growth compared to plain hydrogels (p < 0.009). However, even these surfaces reached a maximum of only 20% confluence. Laminin, fibronectin adhesion-promoting peptide (FAP), and fibronectin/laminin (1:1) tether-modified hydrogels all achieved 100% confluence by the end of the culture period, although the rates at which confluence was reached differed. F-actin staining showed that focal adhesions were formed for the laminin, FAP, and fibronectin/laminin tether-modified surfaces. The results support the hypothesis that tethering certain extracellular matrix proteins and/or peptides to the hydrogel surface enhances epithelial cell growth and adhesion, compared with that seen for proteincoated or plain hydrogel surfaces.